The present invention relates to the field of determining the clotting time of blood samples and more specifically relates to the determination of the clotting time of blood samples from patients receiving heparin treatment, particularly patients that have been administered low to moderate heparin doses, as well as that of patients that have been administered high heparin doses.
The activated clotting time (ACT) assay is a blood test that monitors the effectiveness of heparin dosing. The levels of heparin that the ACT assay is monitoring are generally beyond the range of the activated partial thromboplastin time (APTT) assay. Some APTT assays can monitor plasma heparin levels as high as 1.5 U/mL (which is equivalent to a blood heparin level of about 0.75 U/mL), while the ACT assay can monitor blood heparin levels generally as high as 6 U/mL. The higher end of the blood heparin range (high range; HR) is often used in cardiac pulmonary bypass surgery, while blood levels under 3 U/mL (moderate to low range; LR) but above the effective range of the APTT assay, are used in situations such as cardiac catheterization, extracorporeal membrane oxygenation (ECMO), hemodialysis, and percutaneous transluminal coronary angioplasty (PTCA).
There are various commercially available ACT assays. These typically differ in the specific component that activates clotting, which difference can affect the blood heparin range in which the assay is reliable. Consequently, these assay types are often categorized by the heparin range and corresponding surgical or medical application. One example of such a test is known as the Hemochron(copyright) sold by International Technidyne Corporation. The basic procedure for this test is as follows: A two mL sample of blood is added to a test tube containing dried celite (diatomaceous earth) and a small magnetic bar. The test tube is capped and shaken, then placed in an instrument that starts spinning of the magnetic bar. When the blood begins to clot, the magnetic bar slows or stops spinning. The instrument then notes the length of time till the magnet stopped spinning as the celite ACT time. A variation of this test is known as the Hemochron(copyright) glass ACT assay wherein the test tube is plastic and contains glass particles with a magnetic bar and the sample is only 0.4 mL of blood.
U.S. Pat. No. 4,756,884 and 5,039,617 describe an integrated device containing a predispensed, dry reagent in a capillary track that can be used to measure clotting of blood samples. The entire disclosure of these two patents is incorporated herein by reference. The assignee of the present invention currently markets a system under the designation CoaguChek(trademark) Pro and CoaguChek(trademark) Plus. Certain challenges were presented in designing a reagent for use in this system to measure heparin effectiveness in the range of 3 U/mL and lower. For example, the CoaguChek(trademark) Plus/Pro system should have an assay time of 300 seconds or less, whereas traditional ACT reagents have an assay time of up to 1000 seconds.
In addition, the activators that have been traditionally used in ACT reagents are celite, kaolin or glass particles, which are all insoluble particles. Such activators are typically problematic for the cartridge-reagent system employed in the CoaguChek(trademark) Plus system which uses the blood sample to solubilize the reagent and move the reagent with the blood through the cartridge tracks during the coagulation activation reaction.
Briefly stated, the invention is a method, reagent and test cartridge for the determination of the clotting time of a blood sample by means of a reagent comprising tissue factor and a co-factor. A preferred co-factor is a sulfatide. This invention is preferably used to monitor the effectiveness of heparin therapy in patients that have been administered low to moderate heparin doses that result in blood heparin levels from about 0 to about 3 U/mL. However, it has been surprisingly discovered that the invention can monitor the effectiveness of heparin therapy in patients that have been administered higher heparin doses resulting in blood heparin levels of up to about 6 U/mL. In an alternative embodiment, the sulfatide may be combined with or replaced by a phosphatide.
In accordance with the test cartridge aspect of the invention, the cartridge includes a housing containing an inlet port, a chamber unit and an exit port. The cartridge preferably further comprises a first capillary unit for independently pumping a liquid, such as a blood sample, from said inlet port to said chamber unit. In addition, the preferred cartridge preferably includes a second capillary unit positioned between and operatively connected to said chamber unit and said exit port for independently pumping a liquid from said chamber unit to said exit port. The inlet port, first capillary unit (if present), chamber unit, second capillary unit (if present), and exit port are present in a continuous capillary pathway. Contained within the capillary pathway are a reagent comprising tissue factor and a co-factor, preferably a sulfatide. In an alternative embodiment, a phosphatide may be combined with a sulfatide or a phosphatide may be the sole co-factor.
It should be noted that, as used herein, the terms thromboplastin, tissue factor, and coagulation factor III are all intended to mean the cell-surface protein that initiates coagulation.
It should also be noted that the term sulfatide refers to a class of sulfate derivatives of cerebrosides, which have the following general structure: 
R=fatty acid residue
The term phosphatide refers to a class of glycerol based compounds in which one of the hydroxyl groups is replaced with a phosphoric acid group, and the other hydroxyl groups are replaced with fatty acid esters. Phosphatides are also referred to as phospholipids, phosphoglycerides, and glycerol phosphatides. For more information on phosphatides, see Lehninger, Albert L., Biochemistry, 2nd Edition, Worth Publishers, NY (1975).